The Philadelphia chromosome (Ph.sup.1) is associated with the bulk of chronic myelogenous leukemia (CML) patients (more than 95%), 10-25% of acute lymphocytic leukemia (ALL) patients, and about 2-3% of acute myelogenous leukemias (AML). This abnormal chromosome fuses most of the ABL gene to the 5' two-thirds of the BCR gene. A number of different kinds of evidence support the contention that Bcr-Abl oncoproteins, such as p210 and p185 BCR-ABL, are causative factors in these leukemias.sup.1. The malignant activity is due in large part to the Bcr-Abl protein's highly activated protein tyrosine kinase activity and its abnormal interaction with protein substrates.sup.1,2.
The Bcr-Abl oncoprotein p210 Bcr-Abl is associated with both CML and ALL, whereas the smaller oncoprotein, p185 BCR-ABL, is associated with ALL patients, although some CML patients also express p185.sup.1. Some reports suggest that Bcr-Abl oncoproteins, p210 and p185 BCR-ABL, function at least in part by activating the Ras pathway. The RAS gene is a proto-oncogene involved in controlling normal cell growth. When continuously activated, the Ras protein becomes a potent cancer gene product. Bcr-Abl oncoproteins have been observed by the present inventors and others to perturb normal Ras function.sup.3.
The mechanism by which Bcr-Abl oncoproteins activate p21 Ras involves several factors. One event involves the autophosphorylation of the Bcr-Abl oncoprotein on tyrosine residues within the coding sequence of the first Bcr exon.sup.4. This finding was unexpected, as it had previously been postulated that Bcr-Abl phosphorylates itself on Abl tyrosines, not Bcr tyrosine residues. Several adaptor proteins have been implicated in Ras-activation as well. FIG. 2 lists several such adaptor proteins that contain SH2/SH3 motifs. Such domains have been observed in proteins involved in transmitting growth signals to the nucleus.sup.5.
Grb2 is an adaptor protein that binds to tyrosine phosphorylated receptor proteins. Bcr-Abl induced oncogenesis has also been reported to be mediated by direct interaction with the SH2 domain of the Grb2.sup.3,16. Grb2 also binds mSos1, a GTP exchange factor (see FIG. 3). The latter activates Ras by forming GTP/Ras. GTP/Ras in turn activates Raf, a serine/threonine protein kinase that activates Mek. Mek is a kinase that phosphorylates and activates MAP kinase. The latter is believed to activate and/or regulate various transcription factors (i.e. c-Jun), resulting in cell growth (FIG. 4).
Another protein/protein interaction that has been examined in relation to Bcr-Abl induced malignancy concerns the formation of tetramer structures. In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176-242).sup.6 Domain 1 of Bcr has been shown to form a homotetramer.sup.6 (FIG. 7A and FIG. 7B). Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function and the ability of Bcr-Abl to transform Rat-1 fibroblasts. The Bcr-Abl tetramer activates its inherent Abl tyrosine kinase activity, its actin binding function, and its cellular transformation function.sup.6.
Another peptide that has been implicated in the malignant effects of Bcr-Abl involves Shc.sup.16. Crkl is another adaptor molecule that forms a protein/protein interaction with Bcr-Abl.sup.8,9,17. Still another adaptor molecule that interacts with Bcr-Abl is p120 Ras Gap.sup.7.
Despite the description of events/molecules involved in Bcr/Abl function and pathologies associated with the activities of its gene product therewith, comprehensive strategies for controlling the activation of the Ras oncogene have not been developed. Thus, a need continues to exist in the medical arts for clinical approaches that target effectively and specifically inhibit Ras activation. Such novel techniques would also provide alternatives in inhibiting Philadelphia chromosome-positive cells in tissues, such as in bone marrow.
It is an object of the present invention to define specific peptide sequences from Bcr-Abl that inhibit Bcr-Abl activation of Ras function. Such peptides identify important binding sites on Bcr-Abl for adaptor molecules such as Grb2. A further object of the invention is to identify peptides or a single polypeptide that include important binding sites for other principal targets of Bcr-Abl oncoproteins together with Grb2-directed peptides. A further object of the invention is to provide Bcr as a negative regulator of Bcr-Abl function. Another object of the invention is to provide methods for processing bone marrow, and particularly for enriching Philadelphia chromosome-negative cells in bone marrow culture. Another object of the invention is to provide liposome associated preparations of peptides that mimic binding sites of the principal targets of Bcr-Abl oncoproteins.